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Tail Epidermis Wholemount Data for Cadm1 (MAMQ)

Overview

Skin screen collaboration (Mice were fed on Mouse Breeder Diet (5021, Labdiet) from weaning): Tails were
collected in duplicate from mutant and wild type mice of the same genetic background (≥ 14 weeks). Epidermal
whole mounts were prepared by separating the epidermis from the underlying dermis as an intact sheet following
EDTA digestion. The epidermis was fixed in 4% paraformaldehyde and permeabilised, then stained using a rapid
labelling protocol with antibodies to keratin 14 and 15 (K14, K15) that were directly conjugated with
fluorophores (Alexa 555 (red) for K14 and 488 (green) for K15). Whole mounts were counter-stained with DAPI to
reveal cell nuclei (blue). Preparations’ were imaged using a Leica SP5 Confocal microscope and Maximum Intensity
Projections produced from ~150um Z-series at 4um spacing. DAPI, Alexa 488 and Alexa555 channels were merged.
Images are x10 magnification unless otherwise indicated.

Evaluation of Phenotype

A tick-list was used to record the presence (red cross) or absence (green tick) of a phenotype in the epidermis.

K14 is expressed in basal layer epidermal cells in the interfollicular epidermis (IFE), sebaceous gland (SG) and
hair follicles (HF). DAPI staining provides an indication of cell density and K15 is most highly expressed in the HF
bulge, which is a stem cell reservoir.

In tail epidermis melanocytes reside in the HF and also in the IFE immediately adjacent to the HF (parakeratotic
scale). In some mutant mice pigmentation in the IFE is altered and this is recorded in bright-field images.

In the tick-list, abnormal K14 expression would indicate loss of expression. Abnormal K15 expression indicates
either abnormal bulge morphology or altered K15 expression (for example, reduced in the bulge or up-regulated
outside the bulge). In tail epidermis the HF are normally arranged in groups of three (triplets) and in
staggered rows; abnormalities in HF patterning include changes in number of HF per group and distance between
rows. The hair growth cycle in tail epidermis is less highly synchronised than in the back and the mice analysed
are not closely age-matched. Therefore, detailed evaluation of the hair cycle has not been performed;
nevertheless, obvious disturbance, such as all HF being in catagen, is recorded. The SG are K14-positive
sack-like structures that lie above the bulge; changes in SG size, number or morphology are recorded as
abnormal. Changes in the IFE include hyperproliferation (detected as increased basal layer density and
enveloping of the junction with the HF), hypocellularity and altered pigmentation.

If information or images that you are interested in are not presented, please send
details to MGPenquiries@sanger.ac.uk
and, on a limited, first come first served basis, we will process your request.

The screen is being performed by Emma Heath and Kifayathullah Liakath Ali in the laboratory of Fiona Watt. Our website is www.wattlab.org. We gratefully acknowledge the financial support of
Cancer Research UK, the Medical Research Council, the Wellcome Trust and the European Union. The references
below are the original description of the whole mount tail epidermal preparation (Braun et al., 2003) and a
review on different epidermal stem cell populations (Watt and Jensen, 2009).